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Protochlamydia Naegleriophila Antigen Was Tested with Serum Against All These Different Sp DISPATCHES (7). Ameba fi lled with bacteria exhibiting 3 developmental Protochlamydia stages already described in other Parachlamydiaceae (8) were observed (Figure 1). naegleriophila To measure the serologic differentiation index (SDI) between strain KNic and other Chlamydia-like organisms, as Etiologic Agent we immunized Balb/c mice to produce anti-KNic antibod- ies. Purifi ed Pr. amoebophila (ATCC PRA-7), Simkania of Pneumonia negevensis (ATCC VR-1471), Parachlamydia acanth- Nicola Casson,* Rolf Michel,† Karl-Dieter Müller,‡ amoebae strain Seine, Waddlia chondrophila (ATCC 1470), John David Aubert,* and Gilbert Greub* Neochlamydia hartmannellae (ATCC 50802), Criblamydia sequanensis (CRIB 18), and Rhabdochlamydia crassifi cans Using ameba coculture, we grew a Naegleria endo- (CRIB 01) antigens were tested by micro-immunofl uores- symbiont. Phenotypic, genetic, and phylogenetic analyses cence against mouse anti-KNic antibodies, whereas KNic supported its affi liation as Protochlamydia naegleriophila antigen was tested with serum against all these different sp. nov. We then developed a specifi c diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar la- Chlamydia-like organisms (9). SDIs were calculated as vages, results of this PCR were positive for 1 patient with described (9,10). Serum from mice immunized with KNic pneumonia. Further studies are needed to assess the role showed strong reactivity against autologous antigen (titers of Protochlamydia spp. in pneumonia. of 4,096). Signifi cant cross-reactivity between KNic and Pr. amoebophila (SDI = 7) and P. acanthamoebae (SDI = 10) was observed. Mouse anti-KNic serum did not react ecently, a Naegleria endosymbiont (KNic) was ob- with other Chlamydia-like organisms (Table 1). Because Rserved but remained uncultivable, precluding precise cross-reactivity between members of the order Chlamydi- identifi cation (1). We grew a large amount of strain KNic ales was proportional to the relatedness between each spe- by using Acanthamoeba castellanii, which enabled phe- cies (9), the strong cross-reactivity between KNic and Pr. notypic, genetic and phylogenetic analyses that supported amoebophila supports the affi liation of KNic in the genus its affi liation as Protochlamydia naegleriophila. This new Protochlamydia. ameba-resistant intracellular bacteria might represent a Taxonomic position of KNic was further defi ned by new etiologic agent of pneumonia because it is likely also sequencing 16Sr RNA (rrs, DQ635609) and ADP/ATP resistant to human alveolar macrophages (2,3). Because translocase (nnt, EU056171) encoding genes. The rrs other Parachlamydiaceae were associated with lung infec- was amplifi ed/sequenced using 16SIGF/RP2Chlam prim- tion (4–6), we assessed the role of Pr. naegleriophila in ers (11). The nnt was amplifi ed/sequenced using nntF2p pneumonia by developing a diagnostic PCR and applying (5′-TGT(AT)GAT(CG)CATGGCAA(AG)TTTC-3′) and it to bronchoalveolar lavages. nntR1p (5′-GATTT(AG)CTCAT(AG)AT(AG)TTTTG-3′) primers. Genetic and phylogenetic analyses were conducted The Study by using MEGA software (12). The 1,467-bp rrs sequence KNic growth in A. castellanii was assessed by immu- showed 97.6% similarity with Pr. amoebophila, 91.8%– nofl uorescence (7) with in-house mouse anti-KNic and Al- 93.2% with other Parachlamydiaceae, and 85.7%–88.6% exa488-coupled anti-immunoglobulin antibodies (Invitro- with other Chlamydiales. Based on the Everett genetic gen, Eugene, OR, USA). Confocal microscopy (LSM510; criteria (13), KNic corresponds to a new species within Zeiss, Feldbach, Switzerland) confi rmed the intracellular the Protochlamydia genus because its sequence similarity location of KNic and demonstrated its rapid growth within with Pr. amoebophila is >95% (same genus) and <98.5% A. castellanii. To precisely assess the growth rate, we per- (different species). Phylogenetic analyses of rrs gene se- formed PCR on A. castellanii/KNic coculture by using PrF/ quences showed that KNic clustered with Pr. amoebophila, PrR primers and PrS probe. After 60 hours, we observed with bootstraps of 98% and 95% in neighbor-joining and an increased number of bacteria per microliter of 4 loga- minimum-evolution trees, respectively. The 569-bp nnt se- rithms (online Appendix Figure, available from www.cdc. quence exhibited 91.1% similarity with Pr. amoebophila, gov/EID/content/14/1/167-appG.htm). 65.5%–72.6% with other Parachlamydiaceae, and 55.4%– A. castellanii/KNic and N. lovaniensis/KNic cocul- 72.6% with other Chlamydiales. Phylogenetic analyses of tures were processed for electron microscopy as described nnt sequences showed that KNic clustered with Pr. amoe- *University Hospital Center and University of Lausanne, Lausanne, bophila. On the basis of these analyses, we propose to name Switzerland; †Central Institute of the Federal Armed Forces Medi- strain KNic “Protochlamydia naegleriophila.” cal Services, Koblenz, Germany; and ‡Institut für Medizinische Mi- We then developed a specifi c diagnostic PCR for krobiologie der Universität Essen, Essen, Germany Protochlamydia spp. Primers PrF (5′-CGGTAATACG 168 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 1, January 2008 Protochlamydia naegleriophilIa and Pneumonia Figure 1. Transmission electron microscopy of Protochlamydia naegleriophila. A) Naegleria lovaniensis trophozoite after transfer of endocytobionts; strain KNic (p) from the original host strain showing 15 coccoid bacteria distributed randomly within the cytoplasm of the host ameba. N, nucleus; en, endosome (karyosome) within the nucleus; v, empty food vacuoles. Magnifi cation ×10,500; bar = 1 μm. B) N. lovaniensis trophozoite jammed with numerous endoparasitic stages of Pr. naegleriophila. Magnifi cation ×16,800; bar = 1 μm. C) Enlarged detail of N. lovaniensis trophozoite with intracytoplasmic stages of Pr. naegleriophila. Some stages show binary fi ssion indicated by the fi ssion furrow (arrows). The endoparasites have a wrinkled gram-negative outer membrane rendering a spiny appearance to the endoparasites. Signs of damage are obvious within the cytoplasm of the host ameba. mi, mitochondria. Magnifi cation ×43,500; bar = 0.5 μm. D) Pr. naegleriophila within vacuoles of Acanthamoebae castellanii ameba 2 days postinfection. Elementary bodies (eb) and reticulate bodies (rb) are visible. Elementary bodies harbor a smooth membrane compared with the reticulate bodies, which have a spiny shape. Magnifi cation ×10,000; bar = 2 μm. E) Enlarged detail of A. castellanii trophozoite with intracytoplasmic stages of Pr. naegleriophila 3 days postinfection. Binary fi ssion is observed (di). Magnifi cation ×20,000; bar = 1 μm. F) Crescent body (arrow) within A. castellanii observed 3 days postinfection. Magnifi cation ×20,000; bar = 1 μm. GAGGGTGCAAG-3′) and PrR (5′-TGTTCCGAGGTT a Bland-Altman bias of 0.99 and a limit of agreement of GAGCCTC-3′) as well as probe PrS (5′-TCTGACTGAC 2.87 (Figure 2, panel A). Inter-run variability was low at ACCCCCGCCTACG-3′) were selected. The 5′-Yakima- high concentration, 1.12, 1.71, 0.82, 1.77 cycles for 105, Yellow probe (Eurogentec, Seraing, Belgium) contained 104, 103, 102 copies/μL, respectively. Inter-run variability locked nucleic acids (underlined in sequence above). The was higher at low concentration, 4.22 cycles for 101 cop- reactions were performed with 0.2 μM each primer, 0.1 ies/μL (Figure 2, panel A). Analytical specifi city was tested μM probe, and iTaqSupermix (Bio-Rad, Rheinach, Swit- with bacterial and eukaryotic DNA (Table 2). The PCR zerland). Cycling conditions were as described (14), and slightly amplifi ed DNA from R. crassifi cans, another Chla- PCR products were detected with ABIPrism7000 (Applied mydia-like organism. No cross-amplifi cation was observed Biosystems, Rotkreuz, Switzerland). Each sample was am- with any other bacteria or with human cells. The absence plifi ed in duplicate. Inhibition, negative PCR mixture, and of cross-amplifi cation of P. acanthamoebae is important extraction controls were systematically tested. because this Chlamydia-related bacteria is considered an To allow quantifi cation, a plasmid containing the tar- emerging agent of pneumonia (4–6). get gene was constructed by cloning PCR products into We tested 134 bronchoalveolar lavage samples from pCR2.1-TOPO vector (Invitrogen, Basel, Switzerland). patients with (n = 65) and without (n = 69) pneumonia and Recombinant plasmid DNA quantifi ed using Nanodrop extracted DNA by using a Bio-Rad Tissue Kit. One sample ND-1000 (Witech, Littau, Switzerland) was 10-fold diluted was positive, with 543 and 480 copies/μL. This positive and used as positive controls. result was confi rmed using the 16sigF/16sigR PCR (13), The analytical sensitivity was 10 copy/μL (Figure 2, which targets another DNA segment. This sequence ex- panel A). Intra-run variability was good (Figure 2B) with hibited 99.6% (284/285) similarity with Pr. naegleriophila Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 1, January 2008 169 DISPATCHES Table 1. Antibody titers and serologic differentiation index (SDI) obtained from reciprocal cross-reactions of mouse antiserum with different Chlamydia-like organisms, as determined by immunofluorescence* Antigen titers (SDI) P.a. Strains Pr. Pr. Simkania strain Waddlia Neoclamydia Criblamydia R. naegleriophila amoebophila negevensis Seine chondrophila hartmannellae sequanensis crassificans Pr. 4,096 (0) 256 (7) <4 (14) 512 (10) 4 (20) 16 (14) <4 (20) <4 (13) naegleriophila Pr. 32 (7) 256 (0) <32 (5) 1,024 64 (10) 32 (0) 128 (9) 64 (3) amoebophila
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